Since the catalytic activity of the most commonly measured human serum enzymes is not necessarily relevant to their clinical usefulness in diagnosis and is only a convenient tool for assay, determination of the total mass of each enzyme or isoenzyme released into the circulation by normal cellular activity or damage is more relevant and desirable. Radioimmunoassay (RIA) offers the high degree of specificity and sensitivity required to measure enzyme mass and to simultaneously distinguish isoenzyme species having the same catalytic activity but having different clinical significances. Creatine kinase (CK) was chosen as a model for this approach because of its clinical importance and relatively simple isoenzyme structure and distribution. To develop RIA's for CK, isoenzyme purifications were necessary to provide material for immunization of animals as a source of antibodies for preparing an I125 radiolabeled tracer and for standards. The RIA's developed for these two isoenzymes are applied to the diagnosis and study of diseases of the heart, skeletal muscle, brain, thyroid and other organs and to the detection of heterozygous carriers of muscular dystrophy.